An important form of virus emergence is the cross-species transmission of a virus from its normal animal reservoir to a new animal and/or human host. My studies seek to identify the viral, host, and evolutionary mechanisms that governed the emergence of canine parvovirus (CPV) as a new pandemic pathogen. CPV emerged in dogs in the mid-1970s, and spread world-wide in 1978, and my sponsor (Parrish) laboratory showed that CPV is a variant of feline panleukopenia virus (FPV) and that a key host range barrier to FPV infection in dogs was the cell receptor, transferrin receptor type-1 (TfR). Only a few viral capsid changes allowed CPV to bind the canine TfR and infect canine cells, and this provides an excellent model for defining the molecular interactions that govern cross-species transmission, adaptation, and hence pathogen emergence. It is now also clear that other mammalian hosts can be infected by these parvoviruses and that those hosts likely played roles in the pandemic emergence of CPV. My proposed studies therefore seek to define the range of viral variants found in alternative animal hosts, to determine how these viruses adapt to specific hosts, and also to explain how interactions between the viruses and receptors determine successful cross-species infections. Aim 1: To determine the evolutionary history of CPV emergence as a pandemic pathogen and to define the roles of different hosts in the evolution and cross-species transmission of these viruses. We will isolate and comprehensively characterize viruses from various carnivore species to determine their roles as hosts in parvovirus evolution. Viruses will be characterized by genomic sequencing, phylogenetic analysis, antigenic studies, and replication assays in various host cells. Site-directed mutagenesis of key residue changes in the viruses will be used to test for their effects on viral structures and in controlling host range. Aim 2: To characterize and functionally test the TfRs from various hosts to clarify the role of viral receptor sequence and structural variation in determining susceptibility to parvovirus infection. We will clone and express the TfRs from various carnivore species and characterize receptor-binding properties for viruses (or viral mutants with specific changes) in microfluidic devices using total internal reflection fluorescence (TIRF) microscopy. Variation in host TfR sequences will be examined to identify evidence of positive selection of the receptors during the evolution of the hosts. Viruses grown in different host cells in vitro will be examined for capsid changes that alter receptor binding, where we will use deep genome sequencing to discern the rate and types of viral variation selected. These studies will allow us to uniquely determine the interplay between the viral capsid and host receptors that mediate cross-species transmission and emergence.